【摘要】：應(yīng)激是指機(jī)體在受到各種因素刺激時(shí)所出現(xiàn)的非特異性的全身反應(yīng)。應(yīng)激源可以刺激機(jī)體引發(fā)應(yīng)激反應(yīng)，釋放激素和其他細(xì)胞因子，促進(jìn)機(jī)體適應(yīng)刺激；然而，當(dāng)應(yīng)激過(guò)度或機(jī)體不能適應(yīng)調(diào)節(jié)時(shí)，則會(huì)產(chǎn)生有害的病理生理學(xué)改變。大腦是應(yīng)激作用的主要靶器官之一，動(dòng)物研究已發(fā)現(xiàn)應(yīng)激可以誘導(dǎo)腦部結(jié)構(gòu)和功能的改變，如神經(jīng)細(xì)胞結(jié)構(gòu)的改變，動(dòng)物認(rèn)知、行為的改變等�，F(xiàn)代自然、社會(huì)環(huán)境中，個(gè)體與各種應(yīng)激因素相接觸，如寒冷、酷熱、潮濕等外部環(huán)境理化因素；營(yíng)養(yǎng)缺乏、感覺(jué)剝奪等個(gè)體內(nèi)環(huán)境因素；還有如競(jìng)爭(zhēng)失敗、喪失親人等心理社會(huì)環(huán)境因素。因此研究慢性復(fù)合應(yīng)激對(duì)機(jī)體的影響尤其是對(duì)心理和認(rèn)知功能的影響則顯得極為重要。 以往研究表明：慢性復(fù)合應(yīng)激能夠?qū)C(jī)體多系統(tǒng)尤其是中樞神經(jīng)系統(tǒng)（central never system, CNS）產(chǎn)生損傷作用，其可能機(jī)制包括影響下丘腦-垂體-腎上腺軸（hypothalamic-pituitary-adrenal axis, HPA）、影響谷氨酸（glutamine, Glu）及其受體的釋放、影響突觸結(jié)構(gòu)及其可塑性等。 小膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)的一個(gè)重要的免疫駐留細(xì)胞，已有文獻(xiàn)報(bào)道持續(xù)活化的小膠質(zhì)細(xì)胞能夠產(chǎn)生大量的炎性因子從而對(duì)神經(jīng)元的代謝、關(guān)鍵蛋白的表達(dá)以及神經(jīng)元的存活產(chǎn)生影響。研究顯示內(nèi)外部環(huán)境的微小改變即能刺激并導(dǎo)致小膠質(zhì)細(xì)胞活化從而對(duì)中樞神經(jīng)系統(tǒng)產(chǎn)生影響�；诖宋覀兺茰y(cè)，小膠質(zhì)細(xì)胞活化可能也在慢性復(fù)合應(yīng)激致學(xué)習(xí)記憶損傷中發(fā)揮重要作用。 目的： 通過(guò)建立大鼠慢性復(fù)合應(yīng)激動(dòng)物模型，研究慢性復(fù)合應(yīng)激對(duì)大鼠學(xué)習(xí)記憶能力的影響及其機(jī)制，重點(diǎn)闡明小膠質(zhì)細(xì)胞活化在慢性復(fù)合應(yīng)激所致學(xué)習(xí)記憶損傷中的作用和分子調(diào)控機(jī)制，為慢性復(fù)合應(yīng)激損傷的防治提供一定的實(shí)驗(yàn)依據(jù)。 方法： 1．建立慢性復(fù)合應(yīng)激動(dòng)物模型：40只斷乳的SD雄性大鼠隨機(jī)分為四組：對(duì)照組（Con）；慢性復(fù)合應(yīng)激組（MStress）（禁食/禁水，晝夜顛倒，鼠籠傾斜45度，單籠孤獨(dú)飼養(yǎng)，限制活動(dòng)，4℃冷應(yīng)激，20℃強(qiáng)迫游泳）；對(duì)照給藥組（Con+Mino）；慢性復(fù)合應(yīng)激給藥組（MStress+Mino）。 2．水迷宮檢測(cè)大鼠的空間記憶能力，膜片鉗技術(shù)檢測(cè)大鼠海馬離體腦片LTP誘導(dǎo)水平。 3．免疫組織化學(xué)染色檢測(cè)小膠質(zhì)細(xì)胞活化狀態(tài)；ELISA法檢測(cè)細(xì)胞因子表達(dá)；TUNEL染色檢測(cè)海馬部位神經(jīng)元細(xì)胞凋亡情況；Western Blot檢測(cè)Erk1/2、GluR1等蛋白表達(dá)情況。 結(jié)果： 1．慢性復(fù)合應(yīng)激能夠誘導(dǎo)學(xué)習(xí)記憶能力降低 通過(guò)給予動(dòng)物7種不同的應(yīng)激刺激建立大鼠慢性復(fù)合應(yīng)激的動(dòng)物模型，隱蔽平臺(tái)實(shí)驗(yàn)結(jié)果顯示，慢性復(fù)合應(yīng)激組大鼠到達(dá)平臺(tái)的潛伏期和對(duì)照組相比顯著延長(zhǎng)（P0.05）；空間探索實(shí)驗(yàn)結(jié)果顯示，慢性復(fù)合應(yīng)激組大鼠在目的象限的探索時(shí)間和對(duì)照組相比顯著縮短（P 0.05）；膜片鉗技術(shù)檢測(cè)發(fā)現(xiàn)慢性復(fù)合應(yīng)激組大鼠海馬離體腦片LTP誘導(dǎo)水平顯著低于正常對(duì)照組（P0.05）。 2．慢性復(fù)合應(yīng)激能夠誘導(dǎo)小膠質(zhì)細(xì)胞活化和海馬神經(jīng)元細(xì)胞凋亡 免疫組織熒光化學(xué)法觀察發(fā)現(xiàn)，慢性復(fù)合應(yīng)激組大鼠海馬部位部分小膠質(zhì)細(xì)胞胞體變粗，突觸減少，，呈阿米巴樣，即活化狀態(tài)，海馬神經(jīng)元細(xì)胞凋亡增加。統(tǒng)計(jì)結(jié)果發(fā)現(xiàn)慢性復(fù)合應(yīng)激組小膠質(zhì)細(xì)胞活化數(shù)量和海馬神經(jīng)元細(xì)胞凋亡數(shù)量與對(duì)照組相比顯著增加（P 0.05）。ELISA法檢測(cè)結(jié)果發(fā)現(xiàn)慢性復(fù)合應(yīng)激組炎性因子TNF-α、IL-1β表達(dá)水平顯著高于對(duì)照組（P0.01）。 3．慢性復(fù)合應(yīng)激能夠誘導(dǎo)海馬p-Erk1/2、GluR1表達(dá)水平的降低 Western Blot檢測(cè)結(jié)果發(fā)現(xiàn)，慢性復(fù)合應(yīng)激可導(dǎo)致大鼠海馬p-Erk1/2表達(dá)水平降低，GluR1表達(dá)降低（P 0.05）。 4.抑制小膠質(zhì)細(xì)胞活化能夠降低其相關(guān)炎性因子的表達(dá)并逆轉(zhuǎn)海馬p-Erk1/2、GluR1表達(dá)的改變。 給予米諾環(huán)素干預(yù)后，小膠質(zhì)細(xì)胞活化水平受到顯著抑制（P 0.05），其炎性因子TNF-α、IL-1β表達(dá)水平也顯著降低；經(jīng)Western Blot檢測(cè)發(fā)現(xiàn)p-Erk1/2、GluR1表達(dá)水平均顯著恢復(fù)（P 0.05）； 5.抑制小膠質(zhì)細(xì)胞活化能夠保護(hù)海馬神經(jīng)元細(xì)胞，并提高海馬LTP誘導(dǎo)水平及大鼠的學(xué)習(xí)記憶能力。 給予米諾環(huán)素干預(yù)后，慢性復(fù)合應(yīng)激大鼠海馬神經(jīng)元細(xì)胞凋亡數(shù)量顯著減少；海馬離體腦片LTP誘導(dǎo)水平較單純慢性復(fù)合應(yīng)激組顯著升高（P0.05）；水迷宮實(shí)驗(yàn)檢測(cè)大鼠空間記憶能力顯著高于慢性復(fù)合應(yīng)激組（P0.05）。結(jié)論： 1、慢性復(fù)合應(yīng)激能夠?qū)е麓笫蠛ｑR神經(jīng)元凋亡及突觸可塑性改變，并導(dǎo)致學(xué)習(xí)記憶能力降低。 2、慢性復(fù)合應(yīng)激可能通過(guò)激活小膠質(zhì)細(xì)胞從而誘導(dǎo)海馬神經(jīng)元的凋亡，影響學(xué)習(xí)記憶能力。 3、活化的小膠質(zhì)細(xì)胞可能通過(guò)抑制海馬神經(jīng)元Erk1/2磷酸化水平和GluR1的表達(dá)，從而抑制LTP的誘導(dǎo)水平；同時(shí)通過(guò)抑制海馬神經(jīng)元Erk1/2磷酸化水平誘導(dǎo)海馬神經(jīng)元凋亡，最終影響學(xué)習(xí)記憶能力。
[Abstract]:Stress refers to the non-specific systemic reaction of the body when it is stimulated by various factors. The stress source can stimulate the body to induce stress reaction, release the hormone and other cytokines, and promote the body to adapt to the stimulation; however, when the stress is excessive or the body is unable to adapt to the regulation, harmful pathological and physiological changes can be generated. The brain is one of the main target organs of stress, and animal studies have found that stress can induce changes in the structure and function of the brain, such as changes in the structure of nerve cells, changes in animal cognition, behavior, etc. In the modern natural and social environment, the individual is in contact with various stress factors, such as the physical and chemical factors of the external environment such as the cold, the heat and the humidity, the lack of nutrition, the sensory deprivation, and the like, and also the psychological and social environment factors such as the failure of the competition and the loss of their loved ones. Therefore, it is very important to study the effect of chronic compound stress on the body, especially on the psychological and cognitive function. Previous studies have shown that chronic compound stress can damage the body system, especially the central nervous system (CNS), and its possible mechanism includes the effects of the hypothalamic-pituitary-adrenal axis (HPA), and the release of glutaamine (Glu) and its receptor. Release, Effect of Synaptic Structure and Plasticity The microglia are an important immune-resident cell of the central nervous system, and it has been reported that the sustained activated microglia can produce a large amount of inflammatory factors, which can lead to the metabolism of the neurons, the expression of the key proteins, and the survival of the neurons. The study shows that minor changes in the internal and external environment, i.e., can stimulate and cause the activation of microglia to produce the central nervous system On the basis of this, the activation of microglia may also play a role in learning and memory damage induced by chronic complex stress to act Objective: To study the effect of chronic compound stress on learning and memory of rats and its mechanism by establishing the animal model of chronic compound stress in rats. and can be used for preventing and treating the chronic compound stress injury. certain The experimental results were as follows: 1. Establishment of a chronic compound stress animal model: 40 SD male rats with broken milk were randomly divided into four groups: control group (Con), chronic compound stress group (Mstress) (fasting/ forbidden water, day and night, rat cage). 45 degree oblique, single cage alone, limited activity, cold stress at 4 & deg; C, forced swimming at 20 & deg; C); control administration group (Con + Mino); chronic compound stress administration group (MS stress + Mino. 2. Water maze test of rat's spatial memory ability, patch-clamp technique to detect rat LTP-induced level of the hippocampus from the body. 3. Immunohistochemistry staining to detect the activated state of the microglia; the expression of the cytokines was detected by the ELISA; the apoptosis of the neurons in the hippocampus was detected by the TUNEL staining; and the Western Blot was used to detect Erk1./ 2, GluR1 and other protein expression. Results: 1. Chronic compound stress can induce the ability of learning and memory to reduce the development of rats by giving 7 different stress stimuli to the animal The experimental results of the hidden platform showed that the latency of the rats in the chronic compound stress group was significantly prolonged compared with the control group (P0.05). The experimental results of the space exploration showed that the rats in the chronic compound stress group were in the target quadrant. The time of exploration and the control group were significantly shortened (P 0.05), and the detection of patch clamp technique found that the hippocampus of the rats with chronic compound stress group was isolated from the body of the brain. The P-induced level was significantly lower than that in the control group (P0.05). Combined stress can induce the activation of the microglia and the fluorescent chemical method of the apoptosis in the hippocampal neurons, and the part of the hippocampus in the chronic compound stress group is small. The cell of the glial cell becomes thicker and the synapse is reduced. The results of the statistics show that the number of microglia in the chronic complex stress group and the hippocampal God Compared with the control group, the number of apoptotic cells increased significantly (P 0.05). The level of IL-1 expression was significantly higher in the control group than in the control group (P0.01). The results of the Western Blot test that could induce the level of the expression of p-Erk1/ 2 and GluR1 in the hippocampus found that chronic complex stress could lead to the formation of p-Erk1/ 2 in the rat's hippocampus. Erk1/ 2 expression level decreased, GluR1 expression decreased (P 0.05). 4. Inhibition of microglia activation can reduce its correlation The expression of inflammatory factors and the reversal of the expression of p-Erk1/ 2 and GluR1 in the hippocampus were significantly inhibited (P 0.05), and the level of expression of IL-1 in the inflammatory factors decreased significantly. The expression level of p-Erk1/ 2 and GluR1 was found to be significantly higher than that of P-Erk1/ 2 (P 0.05). It can protect the neurons of the hippocampus and improve the induced level of the LTP in the hippocampus and the learning and memory ability of the rats. The results showed that the level of LTP in the hippocampus from the hippocampus was significantly higher than that of the chronic composite stress group (P0. 05) The experimental results showed that the spatial memory of the rats was significantly higher than that of the chronic compound stress group (P0.05). Chronic compound stress can lead to the changes of neuronal apoptosis and synaptic plasticity in the hippocampus of the rat, and lead to a decrease in learning and memory capacity. 2. The chronic compound stress can induce the apoptosis of the hippocampal neurons by activating the microglia, and the ability of learning and memory. 3. The activated microglia may inhibit the level of Erk1/ 2 phosphorylation and the expression of GluR1 in the hippocampal neurons, thus inhibiting the induction level of LTP.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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